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Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes.

Laulagnier K, Schieber NL, Maritzen T, Haucke V, Parton RG, Gruenberg J - Mol. Biol. Cell (2011)

Bottom Line: Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin.Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3.We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, Switzerland.

ABSTRACT
Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

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Regulation of endo-lysosomal exocytosis is controlled by Rab27a and is BFA-insensitive. (A and B) Living A431 cells overexpressing Rab27a-GFP (green) and Lamp1-cherry (red) were imaged by time-lapse TIRF microscopy at room temperature for 5 min without (A, control, see Supplemental Movie 5) or with 2.5 mM ionomycin (B, 2.5 μM Iono; see Supplemental Movie 1). Snapshots are shown for the indicated time points. Arrows point at a motile vesicle containing Rab27a and Lamp1, and an arrowhead points at an immobile reference in each frame. Bar = 2.5 μm. (C) As in (A), except that cells were pretreated with U18666A as in Figure 2A. Snapshots of Supplemental Movie 4 are shown for the indicated time points. Bar = 5 μm. (D--G) A431 cells were treated or not with 5 μg/ml BFA for 1 h before stimulation. (D) Cells were fixed, permeabilized, and labeled with anti-TGN38 antibody to monitor BFA effect. After BFA treatment, hexosaminidase release (E) was measured as in Figure 1A (means ± SEM of three experiments) or (F) cell surface proteins were biotinylated and analyzed as in Figure 1D. Panel F shows a representative blot and the quantification of three experiments (means ± SEM). (G) After stimulation, total proteins from cell supernatants were precipitated with TCA and blotted against cathepsin D (CD). Three percent of nonstimulated cell lysate was analyzed for comparison. The three cathepsin D isoforms are indicated on the blot.
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Figure 5: Regulation of endo-lysosomal exocytosis is controlled by Rab27a and is BFA-insensitive. (A and B) Living A431 cells overexpressing Rab27a-GFP (green) and Lamp1-cherry (red) were imaged by time-lapse TIRF microscopy at room temperature for 5 min without (A, control, see Supplemental Movie 5) or with 2.5 mM ionomycin (B, 2.5 μM Iono; see Supplemental Movie 1). Snapshots are shown for the indicated time points. Arrows point at a motile vesicle containing Rab27a and Lamp1, and an arrowhead points at an immobile reference in each frame. Bar = 2.5 μm. (C) As in (A), except that cells were pretreated with U18666A as in Figure 2A. Snapshots of Supplemental Movie 4 are shown for the indicated time points. Bar = 5 μm. (D--G) A431 cells were treated or not with 5 μg/ml BFA for 1 h before stimulation. (D) Cells were fixed, permeabilized, and labeled with anti-TGN38 antibody to monitor BFA effect. After BFA treatment, hexosaminidase release (E) was measured as in Figure 1A (means ± SEM of three experiments) or (F) cell surface proteins were biotinylated and analyzed as in Figure 1D. Panel F shows a representative blot and the quantification of three experiments (means ± SEM). (G) After stimulation, total proteins from cell supernatants were precipitated with TCA and blotted against cathepsin D (CD). Three percent of nonstimulated cell lysate was analyzed for comparison. The three cathepsin D isoforms are indicated on the blot.

Mentions: When analyzed by time-lapse video microscopy, Lamp1-containing membranes exhibited high motility across the whole cell cytoplasm (Supplemental Movie 2), whereas in U18666A-treated cells the bulk of Lamp1 was immobile (Supplemental Movie 3) and clustered in perinuclear cholesterol-containing membranes (Figure 2D). Interestingly, despite U18666A treatment, a few Lamp1-positive vesicles remained motile (Supplemental Movie 3) at the cell periphery and did not accumulate cholesterol (Figure 2, D and E). Indeed, peripheral motile endosomes containing Lamp1 were observed by TIRF time-lapse microscopy in U18666A-treated cells (see Figure 5C later in the paper and Supplemental Movie 4) as in untreated controls (see Figure 5A later in the paper and Supplemental Movie 5). Similarly, motile vesicles labeled with LysoTracker have been observed in U18666A-treated cells (Rocha et al., 2009). This small subpopulation of cholesterol-free and motile Lamp1 compartments that we observed could represent the vesicles that are competent for fusion with the plasma membrane.


Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes.

Laulagnier K, Schieber NL, Maritzen T, Haucke V, Parton RG, Gruenberg J - Mol. Biol. Cell (2011)

Regulation of endo-lysosomal exocytosis is controlled by Rab27a and is BFA-insensitive. (A and B) Living A431 cells overexpressing Rab27a-GFP (green) and Lamp1-cherry (red) were imaged by time-lapse TIRF microscopy at room temperature for 5 min without (A, control, see Supplemental Movie 5) or with 2.5 mM ionomycin (B, 2.5 μM Iono; see Supplemental Movie 1). Snapshots are shown for the indicated time points. Arrows point at a motile vesicle containing Rab27a and Lamp1, and an arrowhead points at an immobile reference in each frame. Bar = 2.5 μm. (C) As in (A), except that cells were pretreated with U18666A as in Figure 2A. Snapshots of Supplemental Movie 4 are shown for the indicated time points. Bar = 5 μm. (D--G) A431 cells were treated or not with 5 μg/ml BFA for 1 h before stimulation. (D) Cells were fixed, permeabilized, and labeled with anti-TGN38 antibody to monitor BFA effect. After BFA treatment, hexosaminidase release (E) was measured as in Figure 1A (means ± SEM of three experiments) or (F) cell surface proteins were biotinylated and analyzed as in Figure 1D. Panel F shows a representative blot and the quantification of three experiments (means ± SEM). (G) After stimulation, total proteins from cell supernatants were precipitated with TCA and blotted against cathepsin D (CD). Three percent of nonstimulated cell lysate was analyzed for comparison. The three cathepsin D isoforms are indicated on the blot.
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Related In: Results  -  Collection

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Figure 5: Regulation of endo-lysosomal exocytosis is controlled by Rab27a and is BFA-insensitive. (A and B) Living A431 cells overexpressing Rab27a-GFP (green) and Lamp1-cherry (red) were imaged by time-lapse TIRF microscopy at room temperature for 5 min without (A, control, see Supplemental Movie 5) or with 2.5 mM ionomycin (B, 2.5 μM Iono; see Supplemental Movie 1). Snapshots are shown for the indicated time points. Arrows point at a motile vesicle containing Rab27a and Lamp1, and an arrowhead points at an immobile reference in each frame. Bar = 2.5 μm. (C) As in (A), except that cells were pretreated with U18666A as in Figure 2A. Snapshots of Supplemental Movie 4 are shown for the indicated time points. Bar = 5 μm. (D--G) A431 cells were treated or not with 5 μg/ml BFA for 1 h before stimulation. (D) Cells were fixed, permeabilized, and labeled with anti-TGN38 antibody to monitor BFA effect. After BFA treatment, hexosaminidase release (E) was measured as in Figure 1A (means ± SEM of three experiments) or (F) cell surface proteins were biotinylated and analyzed as in Figure 1D. Panel F shows a representative blot and the quantification of three experiments (means ± SEM). (G) After stimulation, total proteins from cell supernatants were precipitated with TCA and blotted against cathepsin D (CD). Three percent of nonstimulated cell lysate was analyzed for comparison. The three cathepsin D isoforms are indicated on the blot.
Mentions: When analyzed by time-lapse video microscopy, Lamp1-containing membranes exhibited high motility across the whole cell cytoplasm (Supplemental Movie 2), whereas in U18666A-treated cells the bulk of Lamp1 was immobile (Supplemental Movie 3) and clustered in perinuclear cholesterol-containing membranes (Figure 2D). Interestingly, despite U18666A treatment, a few Lamp1-positive vesicles remained motile (Supplemental Movie 3) at the cell periphery and did not accumulate cholesterol (Figure 2, D and E). Indeed, peripheral motile endosomes containing Lamp1 were observed by TIRF time-lapse microscopy in U18666A-treated cells (see Figure 5C later in the paper and Supplemental Movie 4) as in untreated controls (see Figure 5A later in the paper and Supplemental Movie 5). Similarly, motile vesicles labeled with LysoTracker have been observed in U18666A-treated cells (Rocha et al., 2009). This small subpopulation of cholesterol-free and motile Lamp1 compartments that we observed could represent the vesicles that are competent for fusion with the plasma membrane.

Bottom Line: Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin.Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3.We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, Switzerland.

ABSTRACT
Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

Show MeSH
Related in: MedlinePlus